Expression of full length igg and secretion into the culture medium of prokaryotic cells

ABSTRACT

A method for the production of an immunoglobulin or a functional fragment thereof in a prokaryotic host cell comprises transforming the host cell with (a) a first nucleic acid molecule comprising a nucleic acid sequence encoding a V L  and a C L  region and (b) a second nucleic acid molecule comprising a nucleic acid sequence encoding a V H , a C H1 , a C H2  and at least a portion of a C H3  region, The host cell is within culture medium. The host cell is cultured under conditions so as to allow the host cell (a) to express (1) the V L  and a C L  region and (2) the V H , the C H1 , the C H2  and the portion of the C H3  region, and (b) to secrete (a)(1) and (a)(2) to the periplasm of the host cell and thereafter to the culture medium of the host cell. Characteristically, (a)(1) and (a)(2) interact to form the immunoglobulin or functional fragment thereof.

BACKGROUND OF THE INVENTION

The expression of heterologous genes in bacteria is known in the artalmost since modern recombinant DNA technology was available in the1970's. Various strategies have been employed, mostly directed by theneeds, the circumstances and the technological advances available at acertain time. The first recombinant Escherichia coli strain wasgenerated by Cohen et al in 1973 (Proc Natl Acad Sci USA. 1973 November;70(11):3240-3244). In 1977 the first human gene, somatostatin, wasexpressed in prokaryotes (Science, 1977, 198, 1056-1063). Villa-Komaroffet al. (Proc Natl Acad Sci USA, 1978, 75(B), 3727-3731) for the firsttime directed the expression of a heterologous gene, proinsulin, intothe periplasm. The secretion of heterologous genes into the culturemedium of prokaryotes was not accomplished until 1989 (WO91/06655,Schering Corporation). Such secretion however was limited to homomericand relatively short proteins and polypeptides.

The production and secretion of more complex polypeptides, such asmultimeric or heteromeric polypeptides was therefore another obstaclethat had to be overcome. Only in 2002 Simmons et al. described theexpression of full-length immunoglobulins in Escherichia coli and theirsecretion into the periplasm (J Immunol Methods. 2002 May 1;263(1-2):133-47). In 2007 Mazor et al. describe the isolation offull-length IgG antibodies from combinatorial libraries expressed in E.coli (Nat. Biotechnol. 2007 May; 25(5):563-5. Epub 2007 Apr. 15).However, the method described by Mazor et al. still requires thepermeabilization of the outer membrane to release the immunoglobulinsinto the culture medium.

SUMMARY OF THE INVENTION

The present invention overcomes the long felt need to producefull-length immunoglobulins in prokaryotic cells, thereby secreting theimmunoglobulins produced into the culture medium. This enables the easyand convenient purification of functional immunoglobulins directly fromthe cell culture medium of prokaryotic cells.

In one embodiments the invention describes a method for the productionof an immunoglobulin or a functional fragment thereof in a prokaryotichost cell, said method comprising:

-   -   i) transforming said host cell with (a) a first nucleic acid        molecule comprising a nucleic acid sequence encoding a V_(L) and        a C_(L) region and (b) a second nucleic acid molecule comprising        a nucleic acid sequence encoding a V_(H), a C_(H1), a C_(H2) and        at least a portion of a C_(H3) region, wherein said host cell is        comprised within culture medium;    -   ii) culturing said host cell under conditions so as to allow        said host cell (a) to encode (1) said V_(L) and a C_(L) region        and (2) said V_(H), said C_(H1), said C_(H2) and said portion of        said C_(H3) region, and (b) to secrete (a)(1) and (a)(2) to the        periplasm of said host cell and thereafter to the culture medium        of said host cell, wherein (a)(1) and (a)(2) interact to form        said immunoglobulin or functional fragment thereof.

In preferred embodiments of the invention, said light chain of theimmunoglobulin comprises a V_(L) and a C_(L) region. In other preferredembodiments said heavy chain of the immunoglobulin comprises a V_(H), aC_(H1), a C_(H2) and at least a portion of a C_(H3) region. Inalternative preferred embodiments said heavy chain of the immunoglobulincomprises comprises a V_(H), a C_(H1), a C_(H2) and a full-length C_(H3)region. In certain embodiments said immunoglobulin is a functionalfragment of said immunoglobulin. In other preferred embodiments saidimmunoglobulin is a full-length immunoglobulin.

In preferred embodiments of the invention, said immunoglobulin is of theIgG type, most preferably of the IgG1 type.

In certain embodiments of the invention, the first nucleic acidmolecule, which contains a nucleic acid sequence encoding a light chainof an immunoglobulin, further comprises a nucleic acid sequence encodingfor a signal sequence. In other embodiments of the invention, the secondnucleic acid molecule, which contains a nucleic acid sequence encoding aheavy chain of an immunoglobulin, further comprises a nucleic acidsequence encoding for a signal sequence. In most preferred embodiments,both the first nucleic acid molecule comprising a nucleic acid sequenceencoding a light chain of an immunoglobulin and the second nucleic acidmolecule comprising a nucleic acid sequence encoding a heavy chain of animmunoglobulin further comprise a nucleic acid sequence encoding for asignal sequence. In some embodiments these two signal sequences areidentical. In other, preferred, embodiments, the two signal sequencesare different. In certain particular embodiments, the signal sequencecomprised in the second nucleic acid molecule comprising a nucleic acidsequence encoding a heavy chain of an immunoglobulin is the signalsequence of gene phoA of Escherichia coli.

In preferred embodiments of the invention the signal sequences is aprokaryotic signal sequence. Most preferred is a signal sequences ofEscherichia coli, in particular of MalE, LamB, PelB, LivK, TorT, TolB,DsbA, Pac, TorA, PhoA and OmpA, more particularly LamB, PelB, LivK,TorT, TolB, DsbA, Pac, TorA, PhoA and OmpA, and most particularly LamB,PelB, LivK, DsbA, Pac and OmpA. In alternative embodiments, the signalsequences can be a eukaryotic signal sequence. In preferred embodiments,a signal sequence is, e.g., N-terminal with respect to the heavy chainand the light chain.

In certain embodiments of the invention, the method further comprisesthe steps of recovering said immunoglobulin or said functional fragmentthereof from the culture medium. In yet further embodiments, the methodfurther comprises the step of purifying said immunoglobulin or saidfunctional fragment thereof.

In certain preferred embodiments of the invention, the first and thesecond nucleic acid molecules are operably linked to the same promoter.In alternative embodiments, the first and the second nucleic acidmolecules are not operably linked to the same promoter.

In certain preferred embodiments of the invention, the first and secondnucleic acid molecules are comprised within the same vector.

In another embodiment the invention relates to an immunoglobulin or afunctional fragment thereof, produced according to the presentinvention. In preferred embodiments said immunoglobulin is a full-lengthimmunoglobulin. In preferred embodiments said immunoglobulin or afunctional fragment, produced according to the present invention isaglycosylated.

Yet other embodiments of the invention relate to the use of aprokaryotic host cell cell for the production of an immunoglobulin or afunctional fragment thereof, wherein said immunoglobulin or saidfunctional fragment thereof is secreted into the culture medium. Inpreferred embodiments said immunoglobulin or functional fragment thereofcomprises a V_(L) and a C_(L) region and a V_(H), a C_(H1), a C_(H2) andat least a portion of a C_(H3) region.

In preferred embodiments, the prokaryotic host cell used in the presentinvention carries a mutation in at least one protein of the outermembrane. In certain preferred embodiments said host cell carries amutation in the genes minA and/or minB. In most preferred embodiments,said prokaryotic host cell is Escherichia coli, most preferablyEscherichia coli strain WCM104 or Escherichia coli strain WCM105. Inother most preferred embodiments said prokaryotic host cell is producedas described in claim 1 of EP 0338410:

-   -   Process for the preparation of an E. coli strain for protein        secretion of at least 140 mg of protein/I within 48 hours into        the culture medium, characterized in that    -   (a) the structural gene of an exoprotein which is to be        expressed is integrated, in a manner known per se, into a        plasmid which is suitable for expression, to give a hybrid        plasmid,    -   (b) an E. coli strain with a minA and/or minB mutation or an E.        coli strain with a mutation in one protein or in several        proteins of the outer membrane is transformed, in a manner known        per se, with the hybrid plasmid which has been formed,    -   (c) the transformed E. coli strain is subjected, in a manner        known per se, to mutagenesis, and    -   (d) where appropriate is subsequently exposed to substances        acting on the cell wall,    -   (e) the cell material subjected to stages (c) and, where        appropriate, (d) is subjected, in a manner known per se, to a        screening for E. coli mutants with increased protein secretion        compared with the E. coli strain used and    -   (f) where appropriate the E. coli mutant(s) with increased        protein secretion obtained as in stage (e) is (are) rendered        plasmid-free, in a manner known per se.

In particular embodiments, step (c) of claim 1 of EP 0338410 is carriedout via chemical mutagenesis, for example withN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In other particularembodiments, D-cycloserine is used as substance acting on the cell wallin stage (d) in claim 1 of EP 0338410. In other particular embodiments,E. coli DS 410 (DSM 4513) or E. coli BW 7261 (DSM 5231) is used inaccordance with EP 0338410 to generate a prokaryotic cell to be used inthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

A “signal sequence”, “signal peptide” or “secretion signal sequence” asused herein refers to a stretch of amino acids within a polypeptide orprotein which directs said polypeptide or protein, typically a newlysynthesized polypeptide or protein, through a cellular membrane of ahost cell. In prokaryotic cells, signal sequences typically directpolypeptides or proteins through the cytoplasmic membrane into theperiplasmic space. Usually, the signal sequence is present at theN-terminus of a protein or polypeptide and facilitates its transport tothe periplasm or into the culture medium of the host cell. Polypeptidesand proteins comprising a signal sequence are referred to as“preprotein”. The signal sequence is generally removed from theN-terminus of the preprotein by enzymatic cleavage during translocationthrough the membrane, thereby producing the mature protein.

In Escherichia coli, signal sequences typically comprise between about15 to 52 amino acids. Most signal sequences contain a positively chargedN-terminal region (n-region), an apolar hydrophobic core (h-region) anda more polar C-terminal region (c-region). The c-region typicallycontains the cleavage site for signal peptidase. The determination ofsignal sequences is well known to the person skilled in the art. Forexample, signal sequences can be obtained from databases such asSwiss-Prot or GenBank or using annotated genome-wide data sets.

Signal sequence of the present invention may be homologous orheterologous origin. A homologous signal sequence is derived from thesame species as the polypeptide or protein to which it is fused. Incontrast, a heterologous signal sequence is derived from a differentspecies. Any homologous or heterologous signal sequence may be cloned inassociation with a polypeptide or protein which is to be transportedthrough a cellular membrane or into the periplasmic space by the hostcell.

A suitable prokaryotic signal sequence may be obtained from genesencoding, for example, PhoE, MBP, LamB or OmpF OmpA, MalE, PhoA, STIIand other genes.

Preferred signal sequences of the present invention are the followingsignal sequences of Escherichia coli, as well as functional derivativesthereof (all amino acids in one-letter code):

Signal sequence of Amino acid Sequence MalE MKIKTGARILALSALTTMMFSASALALamB MMITLRKLPLAVAVAAGVMSAQAMA PelB MKYLLPTAAAGLLLLAAQPAMA LivKMKRNAKTIIAGMIALAISHTAMA TorT MRVLLFLLLSLFMLPAFS TolBMMNTRVWCKIIGMLALLVWLVSSPSVFAV DsbA MKKIWLALAGLVLAFSASA PacMKNRNRMIVNCVTASLMYYWSLPALA TorA MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQA PhoA MKQSTIALALLPLLFTPVTKA OmpA MKKTAIAIAVALAGFATVAQA

Certain preferred signal sequences are signal sequences of the SECsecretion pathways of Escherichia coli, such as the signal sequences ofMalE, LamB, PelB, LivK, PhoA or OmpA. Other preferred signal sequencesare signal sequences of the SRP secretion pathways of Escherichia coli,such as the signal sequences of TorT, TolB or DsbA. Yet other preferredsignal sequences are signal sequences of the TAT secretion pathways ofEscherichia coli, such as the signal sequences of Pac or TorA.

Other preferred signal sequences are signal sequences of pullulanases(Alpha-dextrin endo-1,6-alpha-glucosidase; EC 3.2.1.41), such as thesignal sequences of the pullulanases of the following species:

Klebsiella aerogenes: MLRYTCHALF LGSLVLLSG Klebsiella pneumoniae:MLRYTRNALV LGSLVLLSG Thermoanaerobacter ethanolicus: M FKRRTLGFLLSFLLIYTAV FGSMPVQFAK A Thermoanaerobacter thermohydrosulfuricus:MFKRRALGFL LAFLLVFTAV FGSMPMEFAK A Thermoanaerobacterthermosulfurugenes: MNKKLFTNRF ISFNMSLLLV LTAVFSSIPL HSVHAThermoanaerobacter saccharolyticum: MYKKLFTKKF ISFVMSLLLV LTAAFSSMPFHNVYA Thermotoga maritime: MKTKLWLLLV LLLSALIFS

Also preferred are the following signal sequences, as well as functionalderivatives thereof (all amino acids in one-letter code):

Heat-stable enterotoxin II of Escherichia coli (STII)

MKKNIAFLLA SMFVFSIATN AYAphoE of Escherichia coli

MKKSTLALWMGIVASASVQAMaltose binding protein (MBP) of Escherichia coli

MKIKTGARIL ALSALTTMMF SASALAAlkaline phosphatase of Escherichia coli

MFSALRHRTA ALALGVCFIL PVHASSPKPG

Penicillinase (EC 3.52.6) of various species

e.g. Staphylococcus aureus: MKKLIFLIVIALVLSACNSNSSHA,Escherichia coli: MKNTIHINFAIFLIIANIIYSSA,Klebiella oxytoca: MLKSSWRKTALMAAAAVPLLLASG, orBacillus cereus: MKNKKMLKIGMCVGILGLSITSLVTFTGGALQVEAKEKTGQVKMurein lipoprotein Lpp of Escherichia coli:

MKATKLVLGA VILGSTLLAGCyclomaltodextrin glucanotransferase of Klebsiella oxytoca:MKRNRFFNTSAAIAISIALNTFFCSMQTIA (SwissProt entry: P08704),as well as functional derivatives thereof.

Also preferred are prokaryotic signal sequences selected from signalpeptides of periplasmic binding proteins for sugars, amino acids,vitamins and ions, including signal peptides such as PelB (Erwiniachrysantemi, Pectate lyase precursor), PelB (Erwinia carotovora, Pectatelyase precursor), PelB (Xanthomonas campestris, Pectate lyaseprecursor), LamB (E. coli, Maltoporin precursor), MalE (E. coli,Maltose-binding protein precursor), Bla (E. coli, Beta-lactamase), OppA(E. coli, Periplasmic oligopeptide-binding protein), TreA (E. coli,periplasmic trehalase precursor), MppA (E. coli, Periplasmic mureinpeptide-binding protein precursor), BglX (E. coli, Periplasmicbeta-glucosidase precursor), ArgT (E. coli, Lysine-arginine-ornithinebinding periplasmic protein precursor), MalS (E. coli, Alpha-amylaseprecursor), HisJ (E. coli, Histidine-binding periplasmic proteinprecursor), XylF E. coli, D-Xylose-binding periplasmic proteinprecursor), FecB (E. coli, dicitrate-binding periplasmic proteinprecursor), OmpA (E. coli, outer membrane protein A precursor) PhoA (E.coli, Alkaline phosphatase precursor), OmpT (E. coli, outer membraneprotein 2b), OmpC (E. coli, outer membrane protein 1b and the 17Kantigen signal sequence of Rickettsia rickettsii.

The signal sequence may also be selected from any of the followingsignal sequences of E. coli, or any functional derivative thereof:

MNKNRGFTPLAVVLMLSGSLALTG MTKHARFFLLPSFILISAALIAGMKMRAVAVFTGMLTGVLSVAGLLSAGAYA MNGSIRKMMRVTCGMLLMVMSGVSQAMKRHLNTCYRLVWNHMTGAFVVASELARARGKRGGVAVALSLAAVTS LPVLAMKTLKNMRRKNLCITLGLVSLLSRGANA MLKKSILPMSCGVLVMVMSGLLDAMKTSSFIIVILLCFRIENVIA MKIRRIVSTIAIALSVFTFAHA MNKTLIAAAVAGIVLLASNAQAMNKAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLVLAVVSTIGNA FAMMYRIRNWLVATLLLLCTPVGA MGSPSLYSARKTTLALAVALSFAWQAPVFAMFKTTLCALLITASCSTFA MKLAACFLTLLPGFAVA MRKITQAISAVCLLFALNSSAVALAMHKFTKALAAIGLAAVMSQSAMA MKKVILSLALGTFGLGMAE MKKSILALSLLVGLSTMSSYAMKKVLIAALIAGFSLSATA MKKLVLAALLASFTFGASA MEFFKKTALAALVMGFSGAALAMRRFLTTLMILLVVLVAGLSAL MKWLCSVGIAVSLALQPALA MRYIRLCIISLLATLPLAVHAMSIQHFRVALIPFFAAFCLPVFA MRLLPLVAAATAAFLVVA MKNTIHINFAIFLIIANIIYSSAMKTFAAYVITACLSSTALAS MNLKKIAIASSVFAGITMALTCHA MIKKASLLTACSTAFSAWAMRKSLLAILAVSSLVFSSASFA MEALGMIETRGLVALIEASDAMVKA MRFLLGVLMLMISGSALAMHKLFYLLSLLMAPFVANA MKHKKKNRLVVAISVALIPYIG MFRLNPFVRVGLCLSAISCAWPVLAMSKRNAVTTFFTNRVTKALGMTLALMMTCQSAMA MIKFSATLLATLIAASVNAMKSKLIILLMLVPFSSFSTE MKSKLIILLTLVPFSSFSTG MKLLKVAAIAAIVFSGSALAMKNKLLFMMLTILGAPGIAAA MNTLLLLAALSSQITFN MKRYLRWIVAAEFLFAAGNLHAMRVKHAVVLLMLISPLSWA MQRLFLLVAVMLLSG MRKLFLSLLMIPFVAKAMVKKAIVTAMAVISLFTLMG MRLRKYNKSLGWLSLFAGTVLLSG MFKSTLAAMAAVFALSALSPAAMAMAVNLLKKNSLALVASLLLAGHVQA MNTIFSARIMKRLALTTALCTAFISAAHAMTQYSSLLRGLAAGSAFLFLFAPTAFA MLLKRRLIIAASLFVFNLSSGFAMKKQTQLLSALALSVGLTLSASFQAVA MFVKLLRSVAIGLIVGAILLVAMPSLRSMNKKVLTLSAVMASMLFGAAAHA MASSALTLPFSRIAHA MRISLKKSGMLKLGLSLVAMTVAASVQAMKKIWLALAGLVLAFSASA MKKGFMLFTLLAAFSGFAQA MKRKVLLIPLIIFLAIAMLKKILLLALLPAIAFA MIKHVLLFFVFISFSVSA MSSKKIIGAFVLMTGILSGMAKVISFFISLFLISFPLYA MSFKKIIKAFVIMAALVSVQAHA MSHYKTGHKQPRFRYSVLARCVAWAMKTILPAVLFAAFATTSAWA MFFNTKHTTALCFVTCMAFSSSSIA MKNITFIFFILLASPLYAMKNITFIFFILLASPLYA MNKVKFYVLFTALLSSLCAHG MNKVKCYVLFTALLSSLYAHGMRRVNILCSFALLFASHTSLA MKFLPYIFLLCCGLWSTISFA MKKTIGLILILASFGSHAMKKTIGLILILASFGSHA MLKKIIPAIVLIAGTSGVVNA MLKKIISAIALIAGTSGVVNAMVMSQKTLFTKSALAVAVALISTQAWS MKKYVTTKSVQPVAFRLTTLSLVMSAVLGSASVIAMSKRNAVTTFFTNRVTKALGMTLALMMTCQSAMA MKKTMMAAALVLSALSIQSALAMKITHHYKSLLSAIISVALFYSAA MKKVTLFLFVVSLLPSTVLA MLNIIHRLKSGMFPALFFLTSASVLAMKKTLLAIILGGMAFATTNASA MNKFISIIALCVFSSYANA MKNKYNLLFFLFLLCYGDVALAMKKLYKAITVICILMSNLQSA MIKKVPVLLFFMASISITHA MNKYPPLLTMLIIGIGSNAVAMTPLRVFRKTTPLVNTIRLSLLPLAGLSFSAFA MLAFIRFLFAGLLLVISHAFAMNKKIHSLALLVNLGIYGVAQA MRLAPLYRNALLLTGLLLSGIAAVQAMARSKTAQPKHSLRKIAVVVATAVSGMSVYAQA MSKRIALFPALLLALLVIVAMSGLPLISRRRLLTAMALSPLLWQMNTAHA MLSTQFNRDNQYQAITKPSLLAGCIALALLPSAAFAMSNKNVNVRKSQEITFCLLAGILMFMAMMVAGRAEAMSYLNLRLYQRNTQCLHIRKHRLAGFFVRLVVACAFA MRNKPFYLLCAFLWLAVSHAMKWCKRGYVLAAILALASATIQA MKRVITLFAVLLMGWSVNAWSFAMKSLFKVTLLATTMAVALHAPITFA MLIIKRSVAIIAILFSPLSTA MQKNAAHTYAISSLLVLSLTGMIKFLSALILLLVTTAAQA MRTLLAILLFPLLVQA MKLAHLGRQALMGVMAVALVAGMSVKSFAMKIKTLAIVVLSALSLSSTAALA MKLKFISMAVFSALTLGVATNAS MRMKKSALTLAVLSSLFSGYSLAMKKLAIIGATSVMMMTGTAQA MKKLAIMAAASMVFAVSSAHA MKFKKTIGAMALTTMFVAVSASAMKKLAIMAAASMVFAVSSAHA MIKSVIAGAVAMAVVSFGAYA MQKIQFILGILAAASSSSTLAMKKTLIALAVAASAAVSGSVMA MKKLAIMAAASMIFTVGSAQA MKRLVFISFVALSMTAGSAMAMIKSVIAGAVAMAVVSFGANA MKKAFLLACVFFLTGGGVSHA MKKTLIALAIAASAASGMAHAMKKTLIALAIAASAASGMAHA MKKTLIALAIAASAASGMAHA MKLKKTIGAMALATLFATMGASAMLKIKYLLIGLSLSAMSSYSLA MKKNLLITSVLAMATVSGSVLA MRIWAVLASFLVFFYIPQSYAMFFGDGGQLLSDKSLTGSAGGGNNRMKFNILPLAFFIG MIKPTFLRRVAIAALLSGSCFSAAAMKSVLKVSLAALTLAFAVSSHA MKLTLKNLSMAIMMSTIVMGSSAMAMFFKKNLTTAAICAALSVAAFSAMA MDCVMKGLNKITCCLLAALLMPCAGHAMKKVLGVILGGLLLLPVVSNA MGYKMNISSLRKAFIFMGAVAALSLVNAQSALAMKKLVLSLSLVLAFSSATAAFA MKKWLLAAGLGLALATSAQA MKQWIAALLLMLIPGVQAMKKSILFIFLSVLSFSPFA MKKSILFIFLSVLSFSPFP MKKNIAFLLASMFVFSIATNAYAMKKLMLAIFISVLSFPSFS MKKTTLALSRLALSLGLALSPLSATAMTIEYTKNYHHLTRIATFCALLYCNTAFS MFSALRHRTAALALGVCFILPVHAMMISKKYTLWALNPLLLTMMAPAVA MKLFKSILLIAACHAAQASA MKLFKSILLIAACHAAQASAMMITLRKLPLAVAVAAGVMSAQAMA MNTKGKALLAGLIALAFSNMALAMKRNAKTIIAGMIALAISHTAMA MMKKIAITCALLSSLVASSVWA MKKAKAIFLFILIVSGFLLVAMKKITGIILLLLAVIILSA MRKRFFVGIFAINLLVG MCGKILLILFFIMTLSAMKKITWIILLLLAAIILAA MKKITGIILLLLAVIILAA MKKITGIILLLLAAIILAAMKKITGIILLLLAVIILAA MKKITGIILLLLAAIILAA MKIKTGARILALSALTTMMFSASALAMKMNKSLIVLCLSAGLLASAPG MNNEETFYQAMRRQGVTRRSFLKYCSLAATSLGLGAGMAPKIAWAMNRRNFIKAASCGALLTGALPSVSHA MMKMRWLSAAVMLTLYTSSSWA MNKTAIALLALLASSASLAMKGRWVKYLLMGTVVAMLAA MFKRRYVTLLPLFVLLAA MKKYLALALIAPLLIS MKAKAILLASVLLVGMARKWLNLFAGAALSFAVAGNALA MKATKLVLGAVILGSTLLAGMKLSRRSFMKANAVAAAAAAAGLSVPGVARA MEFGSEIMKSHDLKKALCQWTAMLALVVSGAVWAMKMRAVAVFTGMLTGVLSVTGLLSAGAYA MDWLLDVFATWLYGLKVIAITLAVIMFMLSTLRRTLFALLACASFIVHA MKLTTHHLRTGAALLLAGILLAG MAYSVQKSRLAKVAGVSLVLLLAAMRFCLILITALLLAG MSAGSPKFTVRRIAALSLVSLWLAG MKKLTVAISAVAASVLMAMSAQAMTRIKINARRIFSLLIPFFFFTSVHA MSVLRSLLTAGVLASGLLWSLNGITATPAAQAMNKGLLTLLLLFTCFAHAQVVDTWQFA MKKTAIAIAVALAGFATVAQA MKVKVLSLLVPALLVAGAANAMMKRNILAVIVPALLVAGTANA MKKLLPCTALVMCAGMA MQTKLLAIMLAAPVVFSSQEASAMRAKLLGIVLTTPIAISSFA MKKIACLSALAAVLAFTAGTSVA MTNITKRSLVAAGVLAALMAGNVALAMFVTSKKMTAAVLAITLAMSLSA MNKNMAGILSAAAVLTMLAGMTMTRLKISKTLLAVMLTSAVATGSAYA MKKRIPTLLATMIATALYSQQGLAMRTLQGWLLPVFMLPMAVYA MKNRNRMIVNCVTASLMYYWSLPALA MQLNKVLKGLMIALPVMAIAAMIKSVIAGAVAMAVVSFGVNNA MKDRIPFAVNNITCVILLSLFCNA MIRKKILMAAIPLFVISGADAMKKIRGLCLPVMLGAVLMSQHVHA MIRLSLFISLLLTSVAVL MKKWFPAFLFLSLSGGNDALAMRLRFSVPLFFFGCVFVHGVFA MVVNKTTAVLYLIALSLSGFIHTFLRA MIKSTGALLLFAALSAGQAIAMRFSRFIIGLTSCIAFSVQA MLIMPKFRVSLFSLALMLAVPFAPQAVAMPRLLTKRGCWITLAAAPFLLFLAAWG MNAKIIASLAFTSMFSLSTLLSPAHAMKKSTLALVVMGIVASASVQA MKKWSRHLLAAGALALGMSAAHA MTALNKKWLSGLVAGALMAVSVGTLAMKAILIPFLSLLIPLTPQSAFA MKQSTIALALLPLLFTPVTKA MNMFFRLTALAGLLAIAGQTFAMRHSVLFATAFATLISTQTFA MKKIRGLCLPVMLGAVLMSQHVHA MIRLSLFISLLLTSVAVLMKKWLPAFLFLSLSGCNDALA MRLRFSVPLFFFGCVFVHGVFA MVVNKTTAVLYLIALSLSGFIHTFLRAMIKSTGALLLFAALSAGQAMA MKRDGAMKITLLVTLLFGLVFLTTVG MFKKGLLALALVFSLPVFAMKVMRTTVATVVAATLSMSAFSVFA MPRSTWFKALLLLVALWAPLSQAMNMKKLATLVSAVALSATVSANAMA MSGKPAARQGDMTQYGGSIVQGSAGVMSGKPAARQGDMTQYGGSIVQGSAGV MSGKPAARQGDMTQYGGSIVQGSAGVMSGKPAARQGDMTQYGGPIVQGSAGV MRKQWLGICIAAGMLAA MRYLATLLLSLAVLITAGMKAFWRNAALLAVSLLPFSSANA MKQLWFAMSLVTGSLLFSANASA MRQVLSSLLVIAGLVSGQAIAMKLKFISMAVFSALTLGVATNASA MVKDIIKTVTFSCMLAGSMFVTCHVCAMAYSQPSFALLCRNNQTGQEFNS MKLKAIILATGLINCIAFSAQA MESINEIEGIYMKLRFISSALAMMTKIKLLMLIIFYLIISASAHA MRRVLFSCFCGLLWSSSGWA MNMTKGALILSLSFLLAAMEKAKQVTWRLLAAGVCLLTVSSVARA MIKRVLVVSMVGLSLVGMARTKLKFRLHRAVIVLFCLALLVALMQGA MKRFSLAILALVVATGAQAMVKSQPILRYILRGIPAIAVAVLLSA MRKLTALFVASTLALGAANLAHA MNKWGVGLTFLLAATSVMAMSLSRRQFIQASGIALCAGAVPLKASA MKNWKTLLLGIAMIANTSFA MAISSRNTLLAALAFIAFQAQAMLKKCLPLLLLCTAPVFA MMNFNNVFRWHLPFLFLVLLTFRAAA MKQALRVAFGFLILWASVLHAMQMKKLLPILIGLSLSGFSSLSQA MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAMRVLLFLLLSLFMLPAFS MNKALLPLLLCCFIFPASG MKRILPLILALVAGMAQAMKRRLWLLMLFLFAGHVPAASA MKQTSFFIPLLGTLLLYG MRCRGLIALLIWGQSVAAMSLTKSLLFTLLLSAAAVQAST MKLSMKSLAALLMMLNGAVMAMKSPAPSRPQKMALIPACIFLCFAALSVQA MSRFQRLTKYVAIGGGAALLLAGAAYLAGAMSRILKRIAAGVVIAGVAALLLAAGGYAAG MMPRIKPLLVLCAALLTVTPAASAMKMKKLMMVALVSSTLALSG MMKTKKLMMVALVSSTLALSG MKHNVKLMAMTAVLSSVLVLSGMKTKKLMMVALVSSTLALSG MKKTLLAAGAVLALSSSFTVNA MKPLHYTASALALGLALMGNAQAMAMAVICLTAASGLTSAYA MAMKKLLIASLLFSSATVYG MGRISSGGMMFKAITTVAALVIATSAMAMKLLQRGVALALLTTFTLASETALA MKLRLSALALGTTLLVG MKKMLLATALALLITGMMKSKMKLMPLLVSVTLISG MKIKNILLTLCTSLLLTNVAAHA MKSVFTISASLAISLMLCCTAQAMKTFFRTVLFGSLMAVCANS MQRRDFLKYSVALGVASALPLWSRAVFA MKTIFRYILFLALYSCCNTVSMYKQAVILLLMLFTASVSA MMTFKNLRYGLSSSVVLAASLFSVLSYA MIKTTPHKIVILMGILLSPSVFAMSKKLGFALSGLMLAMVAGTASA MAKSLFRALVALSFLAPLWLNA MAFKFKTFAAVGALIGSLALVGMPERVLNFRALFLHGLYKMDKPKAYCRLFLPSFLLLSA MSLNFSAFSDVLSPLAECAPTFAMRKIALILAMLLIPCVSFA MSLPSIPSFVLSGLLLI MNSKKLCCICVLFSLLAGMPLRRFSPGLKAQFAFGMVFLFVQPDASA MKKHLLPLALLFSGISPA MKKKVLAIALVTVFTGMGVAQAMKIISKMLVGALALAVTNVYA MHSWKKKLVVSQLALACTLAITSQANA MFKKILFPLVALFMLAGMKLVHMASGLAVAIALAA MKYSSIFSMLSFFILFA MRKFIFVLLTLLLVSPFSFAMNKEQSADDPSVDLIRVKNMLNSTISMS MDLNEASLNAASTRA MQLRKPATAILALALSAGLAQAMELYREYPAWLIFLRRTYAVA MKRVLFFLLMIFVSFGVIA MKKLILIAIMASGLVAMKTNRSLVVIVSLITATLLLTA MFKGQKTLAALAVSLLFTAPVYAMSSNFRHQLLSLSLLVGIAAPWAAFA MPFTSSGGEMSAGKGLLLVICLLFLPLKSAMAMDTVNIYRLSFVSCLVMAMPCAMA MNTFSVSRLALALAFGVTLTA MGKAVIAIHGGAGAISRAMNMKLKTLFAAAFAVVGFCSTA MIMKNCLLLGALLMGFTGVAMA MRYSKLTMLIPCALLLSAMPARHLYFIMTNTWNRLALLIFAVLSLLVAGELQA MITMKKSVLTAFITVVCATSSVMAMKTCITKGIVTVSLTAILLSCSSAWA MYRTHRQHSLLSSGGVPSFIGGLVVFVSAAMKKNIFKFSVLTLAVLSLTA MQYKDENGVNEPSRRRLLKVIGALALAGSCPVAHAMLRNGNKYLLMLVSIIMLTA MYSSSRKRCPKTKWALKLLTAAFLAA MYPVDLHMHTVASTHAMKKSLLGLTFASLMFSAGSAVA MMIKTRFSRWLTFFTFAAAVALA MNMMRIFYIGLSGVGMMFSSMAMKIKSIRKAVLLLALLTSTSFA MSTTHNVPQGDLVLRTLAMPADTNA MKKLTVAALAVTTLLSGSAFAMLELLFLLLPVAAAYG MKILYSFLLLPFFSCA MKKENYSFKQACAVVGGQSAMAMMMKKTLLLCAFLVGLVSSNVMA MKKLALILFMGTLVSFYADA MAMAAVCGTSGIASLFSQAAFAMTVSSHRLELLSPARDAAIA MKFQAIVLASFLVMPYALA MNRIYRVIWNCTLQVFQAMARINRISITLCALLFTTLPLTPMAHA MTKLKLLALGVLIATSAGVAHA MEKNMKKRGAFLGLLLVSAMEIRIMLFILMMMVMPVSYA MLRMTPLASAIVALLLGIEAYAMAAIPWRPFNLRGIKMKGLLSLLIFSMVLPA MKRSIIAAAVFSSFFMSAGVFAMNKYWLSGIIFLAYGLASPAFS MEFYMKAFNKLFSLVVASVLVFSLAGMYDFNLVLLLLQQMCVFLVIAWLMS MPLLKLWAGSLVMLAAVSLPLQA MQMIVRILLLFIALFTFGVQAMRIIFLRKEYLSLLPSMIASLFS MSDLLCSAKLGAMTLALLLSATSLSALAMPDHSLFRLRILPWCIALAMSGSYSSVWA MRKYIPLVLFIFSWPVLCA MIKHLVAPLVFTSLILTGMKIRSLSRFVLASTMFASFTASA MSAFKKSLLVAGVAMILSNNVFA MPKLRLIGLTLLALSATAVSHAMKKFAAVIAVMALCSAPVMA MKLITAPCRALLALPFCYAFS MKIKTILTPVTCALLISFSAHAMKFKTNKLSLNLVLASSLLAASIPAFA MELLCPAGNLPALKA MLKKTLLAYTIGFAFSPPANAMNNVKLLIAGSAFFAMSAQA MRRLPGILLLTGAALVVIA MQCGISDGLPGFSYADADGKFSMKLNIFTKSMIGMGLVCSALPALA MKRLLILTALLPFVGFA MDKSKRHLAWWVVGLLAVAMNRRRKLLIPLLFCGAMLTA MQGTKIRLLAGGLLMMATAGYVQA MNVLRSGIVTMLLLAAFSVGAMKRKLFWICAVAMGMSAFPSFMTQA MKKRVYLIAAVVSGALAVSG MKLRSVTYALFIAGLAAFSTSSLAMLINRNIVALFALPFMASATA MDLLIILTYVAFAWA MKACLLLFFYFSFICQLHGMLLHILYLVGITAEA MMNVSEKMEHFDVAIIGLGPAGSALA MDFSIMVYAVIALVGVAIGWLFAMMRIVTAAVMASTLAVSSLSHA MKPGCTLFFLLCSALTVTTEAHA MKNRLLILSLLVSVPAFAMKASLALLSLLTAFTSHS MKKVLYGIFAISALAATSAWA MVILPLWRRVVKRPALILICLLLQAMIKQTIVALLLSVGASSVFA MKKRHLLSLLALGISTA MKKIIALMLFLTFFAHAMNKYLKYFSGTLVGLMLSTSAFA MMNRVIPLPDEQATLDLGERVAKAMASSSLIMGNNMHVKYLAGIVGAALLMAGCSSS MRLIITFLMAWCLSWGAYA MGILKSLFTLGKSFISQAMFSRVLALLAVLLLSANTWA MKFMKKAKILSGVLLLCFSSPLISQA MNAIISPDYYYVLTVAGQSNAMMMKTVKHLLCCAIAASALISTGVHA MTILSLSRFMLAGVLLASFNASAMKKALQVAMFSLFTVIGFNAQA MKKIALIIGSMIAGGIISAAGFTWVAKA MHRQSFFLVPLICLSSALWAMKKNSYLLSCLAIAVSSA MNKLQSYFIASVLYVMTPHAFA MRPLILSIFALFLAGMKRASLLTLTLIGAFSAIQAAWA MEISFTRVALLAAALFFVG MPTKMRTTRNLLLMATLLGSALFARAMKIILLFLAALASFTVHA MKTIFTVGAVVLATCLLSG MKLMRYLNTKNIIAAGVLLSCMSSIAWGMRYLLIVITFFMGFSSLPAWA MEGSRMKYRIALAVSLFALSAGS MNKVTKTAIAGLLALFAGNAAAMSKRTFAVILTLLCSFCIGQALAGG MPQRHHQGHKRTPKQLALIIKRCLPMVLTGSGMLCTTANAMKRAPLITGLLLISTSCAYA MKALSPIAVLISALLLQGCVAAA MFKRLMMVALLVIAPLSAATAMQTKKNEIWVGIFLLAALLAALFVCLKA MKKINAIILLSSLTSASVFA MTLRKILALTCLLLPMMASAMRYIRQLCCVSLLCLSGSAVA MWKRLLIVSAVSAAMSSMALA MRVIMKPLRRTLVFFIFSVFLCGTVS

Also within the scope of the present invention are all functionalderivatives of the signal sequences described herein. “Functionalderivates” as used in this context refers to any signal sequence whichis based on any of the naturally occurring signal sequences describedherein, but which was intentionally or unintentionally modified, therebystill fulfilling its function of directing polypeptides or proteins intothe prokaryotic periplasmic space or through a cellular membrane.Intentional modifications include purposely introduced amino acidsubstitution, such as by site-directed mutagenesis of the respectivenucleic acid encoding for said amino acids, and purposely introducedinsertions or deletions. Unintentional modifications, such as pointmutations, insertions or deletions, may occur during passage of thesignal sequence on the vector or the genome of the host cell.

Suitable eukaryotic signal sequences may be obtained from genesencoding, for example, gp70 from MMLV, Carboxypeptidase Y, KRE5 protein,Ceruloplasmin precursor, Chromoganin precursor, beta-hexosaminidasea-chain precursor and other genes.

The signal sequences to be employed in the present invention can beobtained commercially or synthesized chemically. For example, signalsequences can be synthesized according to the solid phasephosphoramidite triester method described, e.g., in Beaucage &Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automatedsynthesizer, as described in Van Devanter et. al., Nucleic Acids Res.12:6159-6168 (1984).

A “promoter” as used herein refers to a nucleic acid molecule encoding aregulatory sequence controlling the expression of a nucleic acidmolecule of interest. Promoters which may be used include, but are notlimited to the SV40 early promoter region, the promoter contained in the3′ long terminal repeat of the RSV virus, the herpes thymidine kinasepromoter, the tetracycline (tet) promoter, β-lactamase promoter or thetac promoter. As used herein, “operably linked” means the association oftwo or more DNA fragments in a DNA construct so that the function ofone, e.g. protein-encoding DNA, is affected by the other, e.g. apromoter. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the geneof interest in a cell.

An “immunoglobulin” or “Ig” as used herein refers to a typical proteinbelonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclassthereof), and includes all conventionally known antibodies andfunctional fragments thereof. Immunoglobulins typically comprise fourpolypeptide chains, two identical heavy chains and two identical lightchains. The heavy chains typically comprise a variable region (V_(H))and a constant region (C_(H)), which comprises a C_(H1), a C_(H2) and aC_(H3) region. The light chains typically comprise a variable region(V_(L)) and one constant region (C_(L)). Immunoglobulins of the presentinvention comprise at least a portion of a C_(H3) region. Animmunoglobulin comprising the entire heavy chains and light chains isreferred to as “full-length immunoglobulin”.

A “functional fragment” of an immunoglobulin hereby is defined as afragment of an immunoglobulin that retains an antigen-binding region andwhich comprises at least a portion of a C_(H3) region. A “portion of aC_(H3) region” is hereby defined as comprising at least one amino acidbelonging to said C_(H3) domain of the heavy chain constant region.Typically the heavy chain of a functional fragment comprises a variableregion (V_(H)) and a constant region (C_(H)), which comprises a C_(H1),a C_(H2) and at least a portion of a C_(H3) region. A functionalfragment of an immunoglobulin may also comprise minor deletions oralterations in the V_(H) and/or V_(L) region, provided antigen-bindingis maintained.

An “antigen-binding region” of an antibody typically is found in one ormore hypervariable region(s) of an antibody, i.e., the CDR-1, -2, and/or-3 regions; however, the variable “framework” regions can also play animportant role in antigen binding, such as by providing a scaffold forthe CDRs.

The terms “immunoglobulin” and “antibody” are used interchangeably inthe broadest sense as a protein, which can bind to an antigen,comprising at least an antibody variable region, preferably a V_(H)region and optionally also a V_(L) region. Numerous known antibodysequences are listed, and the conserved structure of antibody variableregions is discussed in Kabat et al. (1991), Sequences of ImmunologicalInterest, National Institutes of Health, Bethesda, Md. A variable regioncomprises three complementarity determining regions (CDRs) and fourframework regions (FRs) arranged in the following order: FR1 CDR1 FR2CDR2 FR3 CDR3 FR4. FRs are conserved in sequence relative to CDRs.

The terms “protein” and “polypeptide” are art recognized and used hereininterchangeably.

A “host cell” as used herein refers to any prokaryotic cell used in thepresent invention to produce immunoglobulins, preferably full-lengthimmunoglobulins, thereby secreting the immunoglobulins produced into theculture medium. Most preferred host cells are prokaryotic cells, evenmore preferred procaryotic cells carrying a mutation in at least oneprotein of the outer membrane. Particularly preferred as host cells areGram-negative prokaryotes, most preferably Escherichia coli. In certainembodiments said Escherichia coli carries a mutation in the gene minAand/or minB. In other embodiments said Escherichia coli is Escherichiacoli strain WCM104. In yet other embodiments said Escherichia coli isEscherichia coli strain WCM105.

BRIEF DESCRIPTION OF FIGURES

FIG. 1:

The IgG construct with Cys (C) to Ser (S) mutations lacking inter chaindisulfide bonds designed for expression in E. coli is shown in panel A.As a contrast the natural IgG construct with disulfide bonds betweenheavy and light chain as well as inter heavy chain disulfide bonds inthe hinge region is depicted in panel B.

FIG. 2:

FIG. 2 shows the architecture of the IgG1 bicistronic expressioncassettes in the expression vectors in detail. Light chain and heavychain are in tandem orientation under control of one Ptac promoter.Translation initiation regions (SD-SEQ) are located in front of thecoding regions of light and heavy chains. Signal sequences directtransport of light and heavy chain into bacterial periplasm.

FIG. 3:

Binding of E. coli WCM105 produced MOR01555 IgG to ICAM-1 coated ontoELISA plates. Serial dilutions of bacterial culture medium were appliedto ELISA plates coated with ICAM-1. Detection of IgG was performed usinga goat-anti human IgG, F(ab′)₂ fragment specific peroxidase conjugatedantibody (Jackson Immuno Research), at a dilution of 1:10.000 in BPBS.

FIG. 4:

Result of Western-Blot analysis of bacterial culture medium, columnflow-through and purified samples of E. coli WCM105 expressions ofMOR01555 IgG and for comparison of a MOR01555_Fab_MH. MOR01555_IgG waspurified via Protein A chromatography, the MOR01555 Fab_MH sample wasapplied to standard IMAC chromatography. Bands representing Ig heavychain, Fab_MH heavy chain fragment and the Ig light chain are indicatedon the right. A triangle (∇) points to an additional band detected inthe purified IgG sample.

EXAMPLES

The present invention can be better understood with reference to thefollowing examples, which are not intended to limit the scope of theinvention as described above.

Example 1 Cloning of IgG Expression Vectors

In order to express full length IgG in E. coli the cDNA sequence codingfor the human IgG1 constant region (NCBI Nucleotide entry: Y14737 (GI:2765424); including CH1, hinge, CH2 and CH3 domains) was de novosynthesized by Geneart AG (Regensburg, Germany) with a codon usageoptimized for expression in E. coli (GeneOptimizer sequence optimizationtechnology of Geneart (Regensburg, Germany)). Moreover, cysteineresidues were mutated to serine residues to avoid disulfide bondformation between the heavy and the light chain and to avoid formationof inter-heavy chain disulfide bonds (FIG. 1).

The optimized heavy chain IgG1 constant region described above wascloned into the vector pEX-FabA-mut-Hind/Xba via the restriction sidesBlpI and HindIII, yielding an IgG expression vector designated pEX_IgGMOR01555. This IgG expression vector contains an expression cassette forMOR01555 human IgG1 lambda with light chain and heavy chain in tandemorientation under control of the P_(tac) promoter. MOR01555 is anantibody specific for ICAM-1.

In order to simplify subcloning of other antibody candidates, vectorbackbone of pEX-FabA-mut-Hind/Xba expression vector was modified inseveral restriction sites. The resulting pEX_MV2_MOR01555_Fab_FS vectorwas used to generate pEX_MV2_MOR01555_IgG1 by subcloning of theoptimized heavy chain IgG constant region described above via BlpI andHindIII.

In the same manner an expression vector construct containing anexpression cassette for MOR03207 was generated, designated pEX_MV2_IgGMOR03207_IgG1. MOR03207 is an antibody specific for lysozyme. FIG. 2shows the architecture of the IgG expression cassette generated. Withexception of VH and VL sequences, the elements of the IgG expressioncassette were identical in pEX_IgG MOR01555, pEX_MV2_MOR01555_IgG1 andpEX_MV2_MOR03207_IgG1. The sequences of all IgG expression cassetteswere confirmed via sequencing using appropriate primers.

The signal sequence fused to the N-terminus of the variable domain ofthe heavy chain was the same for all constructs, i.e. the phoA signalsequence (MKQSTIALALLPLLFTPVTKA).

Various signal sequences were fused to the N-terminus of the variabledomain of the light chain:

Signal sequence of Amino acid sequence MalE (E. coli)MKIKTGARILALSALTTMMFSASALA Lamb (E. coli) MMITLRKLPLAVAVAAGVMSAQAMA PelB(E. coli) MKYLLPTAAAGLLLLAAQPAMA LivK (E. coli) MKRNAKTIIAGMIALAISHTAMATorT (E. coli) MRVLLFLLLSLFMLPAFS TolB (E. coli)MMNTRVWCKIIGMLALLVWLVSSPSVFAV DsbA (E. coli) MKKIWLALAGLVLAFSASA Pac (E.coli) MKNRNRMIVNCVTASLMYYWSLPALA TorA (E. coli)MNNNDLFQASRRRFLAQLGGLTVAGMLGP SLLTPRRATAAQA PhoA (E. coli)MKQSTIALALLPLLFTPVTKA OmpA (E. coli) MKKTAIAIAVALAGFATVAQA

Signal sequences from all three secretion pathways are included in thisselection: SEC (malE, lamB, pelB, livK, phoA, ompA), SRP (torT, tolB,dsbA), TAT (pac, torA).

DNA fragments containing all signal sequences were generated by de-novoDNA synthesis (Geneart, Regensburg, Germany) and cloned into vectorspEX_MV2_MOR03207_IgG1 or pEX_MV2_MOR01555_IgG1 using restriction enzymesEcoRI and EcoRV. The nucleic acid sequences encoding the signalsequences were selected as follows:

Signal sequence of Nucleic acid sequence MalEATGAAAATAAAAACAGGTGCACGCATCCTCGC (E. coli)ATTATCCGCATTAACGACGATGATGTTTTCCG CCTCGGCTCTCGCC LambATGATGATTACTCTGCGCAAACTTCCTCTGGC (E. coli)GGTTGCCGTCGCAGCGGGCGTAATGTCTGCTC AGGCAATGGCT PelBATGAAATACCTATTGCCTACGGCAGCCGCTG (E. coli)GATTGTTATTACTCGCGGCCCAGCCGGCCAT GGCC LivKATGAAACGGAATGCGAAAACTATCATCGCAG (E. coli)GGATGATTGCACTGGCAATTTCACACACCGC TATGGCT TorTATGCGCGTACTGCTATTTTTACTTCTTTTCC (E. coli) CTTTTCATGTTGCCGGCATTTTCG TolBATGAAGCAGGCATTACGAGTAGCATTTGGTT (E. coli)TTCTCATACTGTGGGCATCAGTTCTGCATGC T DsbA ATGAAAAAGATTTGGCTGGCGCTGGCTGGTT(E. coli) TAGTTTTAGCGTTTAGCGCATCGGCG Pac ATGAAAAATAGAAATCGTATGATCGTGAACT(E. coli) GTGTTACTGCTTCCCTGATGTATTATTGGAG CTTACCTGCACTGGCT TorAATGAACAATAACGATCTCTTTCAGGCATCAC (E. coli)GTCGGCGTTTTCTGGCACAACTCGGCGGCTT AACCGTCGCCGGGATGCTGGGGCCGTCATTGTTAACGCCGCGACGTGCGACTGCGGCGCAAG CG PhoA ATGAAACAAAGCACTATTGCACTGGCACTCT(E. coli) TACCGTTGCTCTTCACCCCTGTTACCAAAGC C OmpAATGAAAAAGACAGCTATCGCGATTGCAGTGG (E. coli)CACTGGCTGGTTTCGCTACCGTAGCGCAGGC C

Constructs were transformed into E. coli WCM105 and expression in 20 mlscale was performed as described below. Two parallel expression culturesof each construct were inoculated from corresponding seed cultures.

Example 2 IgG Expression in Escherichia coli

IgG expression vectors (generated as described in Example 1) weretransformed into E. coli WCM105 (E. coli WCM105 can be prepared from E.coli DS410, as described in EP0338410B1, which is hereby incorporate byreference in its entirety) by electroporation. Bacteria were plated onto2xYT or V67 plates containing 10 or 20 μg/ml Tetracycline (Tet) andgrown overnight at 37° C. Seed cultures were inoculated from transformedplates into 2xYT or V67 medium containing 20 μg/ml Tetracycline andgrown overnight at 30° C. and 250 rpm. 20 ml expression cultures in 2xYTor V67 medium supplemented with 20 mg/ml Tetracycline, trace elements,0.3% glucose and 1% lactose were inoculated with pre-culture to a startOD₆₀₀ of 0.1 and grown in 100 ml non-baffled shake flasks at 30° C. and200-250 rpm for 72 h. Cultures were harvested at 5000×g for 10 min andsupernatant sterile filtered. Two parallel expression cultures of eachconstruct were inoculated from a corresponding seed culture.

Example 3 Proof of Concept Via Protein a Affinity Purification

In order to demonstrate that the IgG's were actually secreted into theculture medium of the host cells, the culture supernatant was subjectedto purification via a Protein A affinity column. For this experiment E.coli WCM105 transformed with pEX_MV2_IgG MOR03207_IgG1 was used.

Experimental Procedure:

-   -   After growth of the expression cultures as described in Example        2, 17 ml of the each respective culture media was adjusted with        1.9 ml 10×PBS for purification via a GX-274 Gilson robot    -   0.2 ml Protein A FF (GE Healthcare), packed in 1 ml Varian        plastic columns were used    -   columns were equilibrated with 10 column volumes 1×PBS pH 7.2        and 18.5 ml adjusted supernatants were loaded onto the columns    -   Protein A columns were washed with 10 column volumes 1×PBS    -   Elution was done with 100 mM glycine pH 3 in one 450 μl fraction        adjusted with 50 μl 1M Tris pH 8 after pre-elution with one        column volume    -   fractions of all columns were measured at UV280 nm with a        Nanodrop photometer against elution buffer    -   the column was regenerated with 10 column volumes of 0.5M NaCl        pH 2

A260/A280 Expression rate Experiment No. signal peptid ratio mg/l 1b1malE 0.69 15.0 1b2 malE 0.66 12.1 2b1 lamB 0.73 18.5 2b2 lamB 0.65 20.33b1 pelB 0.61 18.8 3b2 pelB 0.84 22.4 4b1 livK 0.62 12.1 4b2 livK 0.8518.8 6b1 torT 0.96* 27.4* 6b2 torT 0.62 15.6 7b1 tolB 1.10* 32.4* 7b2tolB 1.10* 29.4* 8b1 dsbA 0.71 17.9 8b2 dsbA 0.61 15.9 9b1 pac 0.61 18.89b2 pac 0.60 17.9 10b1  torA 0.67 15.3 10b2  torA 0.93* 19.4* 11b1  phoA1.33* 14.4* 11b2  phoA 0.70 2.4 12b1  ompA 0.68 19.1 12b2  ompA 0.6119.1

As can be seen in Table 1, immunoglobulins could be eluted from theProtein A affinity column with all signal sequences tested. TheA260/A280 ration gives an indication of the contamination of the proteinfractions with nucleic acids. Ratios greater than 0.9 give rise toinaccuracies in the determination of the protein content of therespective fractions and the expression rates calculated therefrom. Suchmeasurements are highlighted with an asterisk (*). Based on the proteincontents of the individual fractions the expression rate wasextrapolated for a culture volume of one litre (last column of Table 1).The expression rates determined are surprisingly high, and higher thanthe values typically received for respective immunoglobulins produced ineukaryotic systems.

The entire experiment was repeated and again two parallel expressioncultures of each construct was inoculated from corresponding seedcultures. Results of this second set of experiments generally confirmedthe expression rates determined in the first set of experiments.

All fractions eluted from the Protein A affinity column were alsosubjected to SDS-PAGE. The heavy and the light chain of theimmunoglobulins are clearly visible. With the exception of somedegradation products of the heavy chain in some fractions, no prominentother bands could be seen on the gel.

In alternative experiments E. coli WCM105 transformed with pEX_MV2_IgGMOR01555 was used. in the following set up:

10 ml plastic columns (Pierce) packed with 1 ml 50% rProtein ASepharose™ Fast Flow (GE) were used for gravity flow purification.Supernatants (5 ml culture medium) were adjusted with 10× Running Buffer(RB) and loaded onto columns equilibrated with 5 column volumes (CV) RB.Column was washed with 10 CV RB and elution done with 5 CV elutionbuffer (EB). Fractions of 250 μl were collected and neutralized with1/10 of neutralization buffer. For regeneration 5 CV were used and thecolumn then re-equilibrated with 10 CV RB. Recipes for buffers areoutlined below:

Running Buffer: 148 mM PBS

Elution Buffer: 100 mM glycine pH 3

Neutralization Buffer: 3 M Tris pH 8 Regeneration Buffer: 0.5 M NaCl pH2

Samples were analysed under denaturing, reducing conditions using 15%Tris-HCl Criterion Precast gel (BioRad, 26-well). Running conditions: 10min 100 V, 50 min 200 V; Running buffer: 25 mM Tris, 192 mM Glycine,0.1% SDS.

The results with the lysozame-binding immunoglobulin MOR03207 could beconfirmed.

In summary, these experiments clearly showed and confirmed thesurprising finding that functional IgG's can be produced in prokaryoticcells, and that said IgG's can be secreted into the culture medium vianumerous signal sequences.

Example 4 ICAM-1 specific Elisa to Confirm the Presence of FunctionalIgG's in Bacterial Culture Medium

Some IgG's were characterized in more detail. In one experiment thepresence of functional IgG's in bacterial culture medium was confirmedvia ELISA. An antibody containing the expression cassette for MOR01555was used in this experiment.

A black 96-well Maxisorp microtiter plate (Nunc) was coated over nightat 4° C. with 50 μl/well of 0.5 μg/ml human ICAM-1-Fc fusion protein(R&D Systems). The ELISA plate was blocked with 100 μl/well PBS+2% BSA(BPBS) for 1-2 h at RT. Purified reference Fab-dHLX, test- and QCsamples were appropriately pre-diluted and applied in 8 serial 2-folddilutions. Per ELISA plate 2 series of reference probe (starting with 20ng/ml for Fab-A dHLX) a High-QC and Low-QC sample and test samples wereapplied. QC samples were spiked into mock control produced in theappropriate medium. The concentration of QC samples was adjusted to thehighest and lowest expected test sample concentration. Samples werediluted in BPBS using polypropylene microtiter plates (Nunc). After 5washing cycles with PBS+0.05% Tween20 (PBST), 50 μl/well of dilutedsamples were transferred to the ELISA plate and incubated for 1-2 h atRT. The plate was washed again as described above and 50 μl/well ofgoat-anti human IgG, F(ab′)₂ fragment specific peroxidase conjugatedantibody (Jackson Immuno Research), diluted 1:10.000 in BPBS was added.After washing, 50 μl/well QuantaBlu peroxidase-substrate solution(Pierce) was added and fluorescence was measured at 320/430 nm Ex/Emusing a SpectraFluor Plus instrument (Tecan).

Data were evaluated with XL-fit software (IDBS, Emeryville, USA) using a4-parameter logistic fit model. Sample concentrations were calculatedform the average of all dilutions within the calibration range. Dilutionlinearity of reference sample and recovery of QC samples were checked inorder to assess data quality of each test run.

This experiment also is a rough measure for the IgG titer in thebacterial supernatant.

Investigation of bacterial culture medium in ELISA revealed specificICAM-1 binding activity which could be titrated by a serial dilution,indicating that a significant amount of functional IgG was secreted intothe bacterial culture medium (FIG. 3).

By using a Fab_dHLX standard of known concentration the IgG titer in thebacterial supernatant could be roughly determined to be about 12.5μg/ml.

In equivalent experiments the presence of functional IgG's isdemonstrated for the other constructs generated in Example 1.

Example 5 Western Blots to Confirm the Production of Full Length IgG's

The presence of full length IgG heavy and light chains in bacterialculture medium was also confirmed via Western Blots. Polypeptides of theculture medium were separated via SDS-PAGE as described in Example 3.Proteins were then blotted on a nitrocellulose membrane for 1 h at 100 Vusing a BioRad Wet-Blot system. The membrane was blocked with 3% milkpowder in TBS containing 0.1% Tween 20. For detection of heavy and lightchains the following antibodies and conditions were applied:

Detection of heavy chain: Sheep anti-human IgG, Fd specific (The BindingSite) 1:10000 and anti-sheep IgG-AP conjugate (Sigma) 1:10.000 as adetection antibody.

Detection of light chain: Anti-human lambda light chain, AP conjugated(Sigma) 1:1000.

The blot was developed using Fast BCIP/NBT substrate (Sigma).

An antibody containing the expression cassette for MOR01555 was used inthis experiment. For a comparison, respective samples of MOR01555 Fab_MHwere investigated in parallel to the IgG samples.

Light chain and heavy chain were sequentially detected using primaryantibodies with respective specificities (FIG. 4).

The Western-Blot in FIG. 4 shows that both Ig light and full lengthheavy chain are present in the MOR01555 IgG sample in bacterial culturemedium at the expected size. However, excess of light chain over heavychain was approx. 10-20 fold in the raw sample.

Though a significant excess of light chain could also be found in theMOR01555 Fab_MH sample, heavy to light chain ratio was much morebalanced than in the IgG sample. This finding indicates, that productionor secretion of full length Ig heavy chain is a somewhat limiting factorand overall IgG yield in supernatant might be drastically increased byimproving secretion of Ig full length heavy chain.

Nearly exclusively light chain was detected in the column flow throughsamples. This finding was expected due to the excess of light chain inthe bacterial culture medium and since purification via Protein A(MOR01555 IgG) and IMAC (MOR01555 Fab_MH) depends on sequences in theheavy chain. In contrast, both purified samples revealed a very balancedamount (˜1:1 relationship) of heavy and light chain indicating thatpairing of heavy and light chain functions normally during secretionprocess or in bacterial culture medium.

Altogether, these data clearly show that full length, functional IgG canbe expressed in prokaryotic cells, such as E. coli WCM105, and issecreted into the bacterial culture medium.

In equivalent experiments the presence of full length IgG heavy andlight chains in bacterial culture medium is demonstrated for the otherconstructs generated in Example 1.

REFERENCES

-   Simmons L C, Reilly D, Klimowski L, Raju T S, Meng G, Sims P, Hong    K, Shields R L, Damico L A, Rancatore P, Yansura D G.-   Expression of full-length immunoglobulins in Escherichia coli: rapid    and efficient production of aglycosylated antibodies.-   J Immunol Methods. 2002 May 1; 263(1-2):133-47.

1. A method for the production of an immunoglobulin or a functionalfragment thereof in a prokaryotic host cell, said method comprising: a.transforming said host cell with (a) a first nucleic acid moleculecomprising a nucleic acid sequence encoding a V_(L) and a C_(L) regionand (b) a second nucleic acid molecule comprising a nucleic acidsequence encoding a V_(H), a C_(H1), a C_(H2) and at least a portion ofa C_(H3) region, wherein said host cell is within culture medium; b.culturing said host cell under conditions so as to allow said host cell(a) to express (1) said V_(L) and a C_(L) region and (2) said V_(H),said C_(H1), said C_(H2) and said portion of said C_(H3) region, and (b)to secrete (a)(1) and (a)(2) to the periplasm of said host cell andthereafter to the culture medium of said host cell, wherein (a)(1) and(a)(2) interact to form said immunoglobulin or functional fragmentthereof.
 2. The method according to claim 1, wherein said heavy chaincomprises a V_(H), a C_(H1), a C_(H2) and a full-length C_(H3) region.3. The method according to claim 1, wherein said immunoglobulin is afull-length immunoglobulin.
 4. The method according to claim 1, furthercomprising the step of recovering said immunoglobulin or said functionalfragment thereof from the culture medium.
 5. The method according toclaim 1, wherein said immunoglobulin is an IgG.
 6. The method accordingto claim 5, wherein said IgG is IgG1.
 7. The method according to claim1, wherein one or more of said first and said second nucleic acidmolecules further comprises a nucleic acid sequence encoding for asignal sequence.
 8. The method according to claim 7, wherein each ofsaid signal sequences is a prokaryotic signal sequences.
 9. The methodaccording to claim 8, wherein one or more of said prokaryotic signalsequences is derived from Escherichia coli.
 10. The method according toclaim 9, wherein said prokaryotic signal sequence is selected from thegroup consisting of the signal sequences of MalE, LamB, PelB, LivK,TorT, TolB, DsbA, Pac, TorA, PhoA and OmpA.
 11. The method according toclaim 7, wherein either or both of said signal sequence is N-terminalwith respect to the heavy chain and the light chain.
 12. The methodaccording to claim 4, further comprising the step of purifying saidimmunoglobulin or said functional fragment thereof.
 13. The methodaccording to claim 1, wherein said first and second nucleic acidmolecules are operably linked to the same promoter.
 14. The methodaccording to claim 1, wherein said first and second nucleic acidmolecules are not operably linked to the same promoter.
 15. The methodaccording to claim 1, wherein said first and second nucleic acidmolecules are within the same vector.
 16. An immunoglobulin or afunctional fragment thereof produced according to claim
 1. 17. Animmunoglobulin or a functional fragment thereof produced according toclaim 1, wherein said immunoglobulin or said functional fragment thereofis aglycosylated.
 18. The method of claim 1 wherein said immunoglobulinor a functional fragment thereof, wherein said immunoglobulin or saidfunctional fragment thereof is secreted into the culture medium.
 19. Themethod of claim 1, wherein said immunoglobulin or functional fragmentthereof comprises a V_(L) and a C_(L) region and a V_(H), a C_(H1), aC_(H2) and at least a portion of a C_(H3) region.
 20. The method ofclaim 18, wherein said prokaryotic host cell carries a mutation in atleast one protein of the outer membrane.
 21. The method of claim 18,wherein said prokaryotic host cell is Escherichia coli.
 22. The methodof claim 21, wherein said Escherichia coli carries a mutation in thegene minA and/or minB.
 23. The method of claim 20, wherein saidEscherichia coli is Escherichia coli strain WCM104 or Escherichia colistrain WCM105.